KR101756682B1 - Cedecea lapagei strain having nitrogen fixation activity, microbial agent containing the same and biofertilizer containing the same - Google Patents

Abstract

The present invention relates to a process for the preparation of Cedecea lapagei ), a microorganism preparation containing the microorganism, and a biological fertilizer containing the microorganism, and a novel Cedecea lapagei ), a microorganism preparation containing the microorganism, and a biological fertilizer.

Description

TECHNICAL FIELD [0001] The present invention relates to a Cedexa rapaeae strain having nitrogen fixation ability, a microorganism preparation containing the same, and a biopreparative fertilizer containing the microorganism preparation and a biopreparative fertilizer containing the same. ≪ Desc / Clms Page number 1 >

The present invention relates to a process for the preparation of Cedecea lapagei ), a microorganism preparation containing the microorganism, and a biological fertilizer including the microorganism preparation.

Globally, over the past decade, we have been using chemical pesticides for disease, pest and weed control, along with a large amount of chemical fertilizer each year to increase the yield of crops such as grains, vegetables, and fruit trees. Such continuous use of chemical fertilizers and pesticides has continuously raised problems such as soil acidification, loss of power, environmental pollution and ecosystem destruction, lethal toxicity, pest resistance and resistance to pesticides in weeds.

In order to solve these problems at home and abroad, efforts are being made to reduce the use of chemical fertilizers and pesticides. In particular, in 1999, the Korean government enacted the Environmentally Friendly Farming Act in order to reduce the use of chemical fertilizers and pesticides by about 40% have. At the same time, the public is increasingly demanding safer food due to improved living standards, and agriculture-friendly agriculture such as organic farming and natural farming methods that aim to preserve agricultural production environment and natural ecosystem is rapidly expanding, And studies on the development of fertilizers and biopesticides using microorganisms that can replace pesticides have been rapidly increasing. The microbial agents thus developed are formulated after large-scale cultivation and are used as a microbial fertilizer or as a pest control agent by soil treatment with spraying or spraying on the leaves.

On the other hand, nitrogen (N), which is an important plant nutrient, is largely retained by application of chemical fertilizer, and it causes difficulty in plant growth when nitrogen is deficient. Over-exploitation of chemical fertilizers to increase yields is contrary to the principle of sustainable agriculture that seeks high productivity with low input, and can also be an environmental problem (Shantharam, S. and AK Mattoo, 1997. Plant Soil 　 194, 205-216). Despite the recognition of the shortcomings of chemical fertilizers, there is little known genetic potential for maximum crop harvesting (Kennedy et al. 2004. Soil Biol . Biochem . 36, 1229-1244). Biological Nitrogen Fixation (BNF) using fixed atmospheric nitrogen, one of the complex interactions between nitrogen-fixing bacteria and higher plants, can help maintain optimum yields with nutrient supply, especially nitrogen supply .

Nitrogen-fixing bacteria are capable of promoting plant growth in environments where nitrogen is not available, although C is sufficient because plants have the ability to convert N ₂ in the atmosphere to ammonia available. Nitrogen-fixing bacteria can be placed under the Plant Growth Promoting Rhizobacteria (PGPR), a group of bacteria that has beneficial effects on plant growth and yield, because it promotes plant growth by another mechanism besides nitrogen fixation to be. In addition to reducing the use of nitrogen fertilizers, exploiting the interaction of plants and nitrogen-fixing bacteria can increase the efficiency of the fertilizer used (Okon, Y. and CA Labandera-Gonzolez 1994. Soil Biol . Biochem . 26, 1591-1601).

One aspect of the present invention is to provide a microorganism preparation containing the same, a microorganism preparation containing the same, a microorganism preparation containing the microorganism preparation, The present invention provides a biocidal fertilizer containing the same.

One embodiment of the present invention is a method of treating a plant having a nitrogen fixation ability, such as Cedecea lapagei ).

The strain may be selected from the group consisting of Cedecea lapagei ) MK7 strain (KACC 81003BP).

Another embodiment of the present invention provides a microorganism preparation comprising, as an active ingredient, a Cedecea lapagei strain having nitrogen fixing ability or a culture thereof.

The strain may be selected from the group consisting of Cedecea lapagei ) MK7 strain (KACC 81003BP).

The microorganism preparation is preferably a microorganism preparation for biological fertilizer.

Another embodiment of the present invention provides a biological fertilizer comprising the microbial agent.

The microbial agent may be selected from the group consisting of Cedecea lapagei ) MK7 strain (KACC 81003BP).

The nitrogen fixability of the present invention, Cedecea lapagei ) can be used as a biological fertilizer in place of chemical fertilizer because of its high nitrogen fixation ability. Therefore, Cedecea, which is an environmentally friendly plant and cultivable crop, lapagei ) and a microorganism preparation containing the same.

Figure 1 is a graphical representation of the Cedecea lapagei ) MK7 strain (KACC 81003BP).

Hereinafter, preferred embodiments of the present invention will be described. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. Further, the embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.

One embodiment of the present invention is a method of treating a plant having a nitrogen fixation ability, such as Cedecea lapagei ).

At this time, the above-mentioned Cedecea lapagei ) strain has been deposited with the Cedecea lapagei ) MK7 strain (KACC 81003BP), and the above-mentioned Cedecea lapagei ) strain was deposited with the Environmental Microorganisms Bank of Korea under the name of Cedecea deposited with Accession No. KEMB 1501-001 lapagei ) strain (KEMB 1501-001).

The above-mentioned Cedecea lapagei strain, particularly the Cedecea lapagei strain MK7 (KACC 81003BP), can help to maintain optimal yields of nutrients, especially nitrogen, by feeding nitrogen to plants through nitrogen fixation. The above-mentioned Cedecea lapagei strain can convert nitrogen (N ₂ ) in the atmosphere to ammonia through the nitrogen fixation ability, so that the carbon source (C) is sufficient but it can promote the growth of plants even in an environment lacking nitrogen source (N).

Specifically, the above-mentioned Cedecea lapagei ) The MK7 strain (KACC 81003BP) preferably contains the NifH gene as a gene exhibiting nitrogen fixation ability, and preferably is a strain capable of fixing nitrogen.

Another embodiment of the present invention provides a microorganism preparation comprising, as an active ingredient, a Cedecea lapagei strain having nitrogen fixing ability or a culture thereof.

At this time, the strain was found to be Cedecea lapagei ) MK7 strain (KACC 81003BP).

In addition, the microorganism preparation is preferably a microorganism preparation for biological fertilizer that can be applied to biological fertilizer to replace chemical fertilizer, and the microorganism preparation can be formulated in a conventional manner, for example, in the form of a dry powder It can be produced in liquid fertilizer form. However, the formulation is not particularly limited.

In the case of the liquid fertilizer type, it can be used by being mixed with the soil by spraying on the soil, and in the form of a dry powder, it can be sprayed on the soil in the form of fertilizer. In addition, the microbial agent may be mixed with a carrier, a corrosive soil, and the like, but is not limited thereto.

Meanwhile, the microorganism preparation is applied to various plants and can be utilized as a biological fertilizer. In this case, the plant can be applied to a microorganism such as corn, carrot, cabbage, potato, etc., Ammonia nitrogen can be supplied to the plant by fixing the nitrogen in the atmosphere by the agricultural method, and the amount of the harvest can be increased.

Another embodiment of the present invention provides a biological fertilizer comprising the microbial agent. At this time, the microbial agent may be selected from the group consisting of Cedecea lapagei ) MK7 strain (KACC 81003BP), and the above-mentioned Cedecea lapagei MK7 strain (KACC 81003BP) can fix ammonia nitrogen to the plant by fixing the nitrogen in the atmosphere.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by the following Examples.

< Example  And Comparative Example >

1. Soil and medium composition

The medium was prepared by mixing malic acid medium and soil extract 0.1%. Specifically, the malic acid medium contained 3.2 g of malic acid, 3.0 g of KH ₂ PO ₄ , MgSO ₄ 0.1 g _, CaCl ₂ 5 mg of monosodium glutamate, 0.1 g of trace elements (1X) and 1 ml of vitamin solution were mixed and sterilized at a final pH of 6.3.

At this time, the trace elements were extracted from the soil using shaking distilled water and ethanol for 3 days with shaking culture (30 ° C., 120 rpm). The soil extract was filtered using a membrane filter (pore diameter: 0.22 μm) After the filter was applied.

2. Nitrogen Fixed Microbial Isolation

Nitrogen - fixing microorganisms were isolated from the soil collected from Mungyeong, Gyeongsangnam - do and Yeoncheon, Chungcheongbuk - do. At this time, the strain was separated using a continuous dilution method and a flat plate smear method. Specifically, 1 g of soil was added to 9 ml of first distilled water, vortexed for 1 hour and then diluted to 10 ^-7 . Samples diluted to 10 ^-5 , 10 ^-6 , and 10 ^-7 were plated on a nitrogen-free medium using a flat plate coating method, and cultured at 28 ° C for 3 days.

3. Nitrogen Fixability  Measure

Acetylene Reduction Assay (ARA) was used to measure nitrogen fixation. The acetylene reduction method is a method of indirectly measuring the activity of a nitrogenase, which is an enzyme capable of reducing a substance having a triple bond such as N ₂ gas or acetylene, commonly having nitrogen fixing microorganisms, The nitrogen fixation ability can be measured by measuring the amount of ethylene produced when acetylene is reduced to ethylene by the enzyme.

The amount of change in acetylene and ethylene was measured by a GC-TCD (Gas Chromatography-Thermal Conductivity Detector). The conditions of the GC-TCD are shown in Table 1 below.

Oven Inlet Detector Carrier gas 60 캜, 4 minutes, holding (holding) 250 ℃ 250 ℃ Helium (He) 200 占 폚, 1 minute, Holding (60 占 폚 / min) Split ratio
50: 1

4. Smoothness anaerobe  Under nitrogen conditions Fixability  Measure

As a result of measuring the nitrogen fixation ability by a acetylene reduction method using a single strain of Rhizobium sp., Which is a typical nitrogen-fixing strain, Rhizobium sp., As shown in Table 2, in the case of strains of Comparative Examples 1 to 11, The amount of acetylene was reduced, but the removal rate was very low, ranging from 2.11 to 16.63%, and the formation of ethylene was not confirmed.

division Strain Acetylene (ml) Ethylene production (ml) 0 days
Average (standard) 5 days
Average (standard) Removal rate (%) Comparative Example 1 Rhizobium soli R001-001 4.99 (0.56) 4.16 (0.15) 16.63 0 Comparative Example 2 Rhizobium monogolense R001-002 5.00 (0.24) 4.90 (0.53) 1.90 0 Comparative Example 3 Rhizobium gallicum R001-003 5.44 (0.27) 4.76 (0.17) 12.50 0 Comparative Example 4 Rhizobium loessense R001-004 5.24 (0.24) 5.00 (0.40) 4.58 0 Comparative Example 5 Rhizobium mesosinicom R001-005 5.72 (0.70) 4.88 (0.40) 14.69 0 Comparative Example 6 Rhizobium fabea R001-006 5.21 (0.31) 5.10 (0.56) 2.11 0 Comparative Example 7 Rhizobium fleshy R001-007 4.83 (0.00) 4.44 (0.00) 8.00 0 Comparative Example 8 Rhizobium sullae R001-008 5.29 (0.46) 4.93 (0.31) 6.81 0 Comparative Example 9 Rhizobium pisi R001-009 5.22 (0.57) 5.02 (0.53) 3.74 0 Comparative Example 10 Rhizobium alamii R001-010 5.26 (0.52) 5.04 (0.38) 4.09 0 Comparative Example 11 Rhizobium yanglingense R001-011 5.22 (0.74) 4.83 (0.49) 7.48 0

On the other hand, the strain isolated from the soil was used to confirm whether or not ethylene was produced using the acetylene reduction method. As a result, as shown in Table 3, Cedecea lapagei ) MK7 strain (KACC 81003BP).

division Strain Acetylene (ml) Ethylene production (ml) 0 days 5 days Removal rate (%) Comparative Example 12 Paenibacillus sonchi MK5 6.37 5.34 16.11 0 Example 1 Cedecea lapagei MK7 (KACC 81003BP) 6.22 2.63 57.73 1.08

As shown in Table 3, the Cedecea of Example 1 lapagei ) MK7 strain (KACC 81003BP) was found to have a high removal rate of acetylenes of 57.73% by the acetylenic reduction method, in particular 1.08 ml of ethylene was produced, indicating that the strains have excellent nitrogen fixation ability .

5. 16S rRNA  Identification of Nitrogen Fixing Microorganisms by Nucleotide Sequence

On the other hand, as a strain having nitrogen fixability identified in Table 3, Cedecea To isolate MK7, a single isolated strain was submitted to Macrogen, Inc. for analysis of the base sequence of the 16S rRNA gene, and the nucleotide sequence was analyzed using the commonly used primers 785F and 907R. The nucleotide sequence obtained from the analysis was confirmed by using EZbiotexon, and as a result, the Cedecea lapagei) MK7 was characterized by the degree of similarity between the sede Seah Rapa Gay (Cedecea lapagei) GTC ^T 346 and 98.36%.

Polymerase chain reaction (PCR) was performed to identify Nitrogen fixing gene (Nif gene). Cedecea The PCR was performed using primer NifH (F-5 'CGTTTT ACGGCAAGGGCGGTATCGGCA-3', R-5 'TCCTCCAGCTCCTCCATGGTGATCGG-3') after extracting DNA from lapagei MK7 strain (KACC 81003BP). After pre-denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 56 ° C for 40 seconds, and extension at 72 ° C for 30 seconds After 30 cycles of repetition, they were stored at 4 ° C after post-extensiong at 72 ° C for 7 minutes.

The byproducts obtained from the polymerase chain reaction were identified by electrophoresis in 1% agarose gel (1% agarose gel) and 1X TAE buffer for 20 minutes. As a result, as shown in Fig. 1, the target gene NifH (arrow) was amplified by about 230 bp using Cedecea lapagei ) MK7 strain (KACC 81003BP) (M is a 1 kb plus marker and MK7 is Cedecea lapagei ) DNA was extracted from MK7 strain (KACC 81003BP), and the byproducts were electrophoresed after PCR using primer NifH).

National Institute of Agricultural Science KACC81003BP 20150629

<110> KYONGGI UNIVERSITY INDUSTRY & ACADEMIA COOPERATION FOUNDATION <120> CEDECEA LAPAGEI STRAIN HAVING NITROGEN FIXATION ACTIVITY,          MICROBIAL AGENT CONTAINING THE SAME AND BIOFERTILIZER CONTAINING          THE SAME <130> DPP-2015-1310-KR <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1471 <212> RNA <213> Unknown <220> <223> Cedecea lapagei <400> 1 tttacactgt gggcacacgt atcacacgca catctagcgc tatcgcacaa gaagctggtt 60 tttgggtgac gagcggcgga cgggtgagta atgtctggga aactgcctga tggaggggga 120 taactactgg aaacggtagc taataccgca taacgtcgca agaccaaaga gggggacctt 180 cgggcctctt gccatcagat gtgcccagat gggattagct agtaggtggg gtaacggctc 240 acctaggcga cgatccctag ctggtctgag aggatgacca gccacactgg aactgagaca 300 cggtccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga 360 tgcagccatg ccgcgtgtat gaagaaggcc ttcgggttgt aaagtacttt cagcgaggag 420 gaaggcattg tggttaataa ccgcagtgat tgacgttact cgcagaagaa gcaccggcta 480 actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc 540 gtaaagcgca cgcaggcggt ctgtcaagtc ggatgtgaaa tccccgggct caacctggga 600 actgcattcg aaactggcag gctagagtct tgtagagggg ggtagaattc caggtgtagc 660 ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacaaaga 720 ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780 ccgtaaacga tgtcgacttg gaggttgttc ccttgaggag tggcttccgg agctaacgcg 840 ttaagtcgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctact 960 cttgacatcc acggaattta gcagagatgc tttagtgcct tcgggaaccg tgagacaggt 1020 gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc 1080 aacccttatc ctttgttgcc agcggttcgg ccgggaactc aaaggagact gccagtgata 1140 aactggagga aggtggggat gacgtcaagt catcatggcc cttacgagta gggctacaca 1200 cgtgctacaa tggcgcatac aaagagaagc gacctcgcga gagcaagcgg acctcataaa 1260 gtgcgtcgta gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta 1320 atcgtagatc agaatgctac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accatgggag tgggttgcaa aagaagtagg tagcttaacc ttcgggaggg cgcttaccac 1440 tttgtgattc atgactgggg tgaagtcgta a 1471

Claims (7)

Cedecea lapagei strain MK7 (KACC 81003BP) with nitrogen fixability.
delete A microorganism preparation containing as an active ingredient Cedecea lapagei strain MK7 (KACC 81003BP) or a culture thereof having nitrogen fixation ability.
delete The microbial agent according to claim 3, wherein the microbial agent is a microbial agent for biological fertilizer.
A biological fertilizer comprising the microbial agent of claim 3.
delete

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